Transcriptomic signature, bioactivity and safety of a non-hepatoxic analgesic generating AM404 in the mid-brain PAG region

The safe and effective management of pain is a critical healthcare and societal need. The potential for misuse and addiction associated with opioids, nephrotoxicity, and gastrointestinal damage from chronic non-steroidal anti-inflammatory drug (NSAID) use, as well as acute liver injury from paracetamol (ApAP) overdose, are unresolved challenges. To address them, we developed a non-opioid and non-hepatotoxic small molecule, SRP-001. Compared to ApAP, SRP-001 is not hepatotoxic as it does not produce N-acetyl-p-benzoquinone-imine (NAPQI) and maintains hepatic tight junction integrity at high doses. SRP-001 has comparable analgesia in pain models, including the complete Freund’s adjuvant (CFA) inflammatory von Frey. Both induce analgesia via N-arachidonoylphenolamine (AM404) formation in the midbrain periaqueductal grey (PAG) nociception area, with SRP-001 generating higher amounts of AM404 than ApAP. Single-cell transcriptomics of PAG uncovered that SRP-001 and ApAP also share modulation of pain-related gene expression and cell signaling pathways, including the endocannabinoid, mechanical nociception, and fatty acid amide hydrolase (FAAH) pathways. Both regulate the expression of key genes encoding FAAH, 2-AG, CNR1, CNR2, TRPV4, and voltage-gated Ca2+ channel. Interim Phase 1 trial results demonstrate SRP-001’s safety, tolerability, and favorable pharmacokinetics (NCT05484414). Given its non-hepatotoxicity and clinically validated analgesic mechanisms, SRP-001 offers a promising alternative to ApAP, NSAIDs, and opioids for safer pain treatment.


Introduction
Worldwide, pain affects 27% of adults 1 , and given its high prevalence and disability sequelae, it is a global health burden. In the United States (U.S.), pain affects more adults than diabetes and cancer combined, with an estimated cost of $635 billion/year to the healthcare system 2 . Current medications are either addictive (e.g., opioids) or cause harm to the liver (e.g., acetaminophen/paracetamol or ApAP) or kidney (e.g., non-steroidal anti-in ammatory drugs; NSAIDs). Although ApAP is an effective pain reliever in various acute and chronic pain conditions 3-10 , its narrow therapeutic index due to the risk of hepatotoxicity limits its clinical utility. ApAP hepatotoxicity remains the most common cause of acute liver failure in the U.S. 11 and the United Kingdom (U.K.) 12 . Annually, ~30,000 patients are hospitalized for ApAP hepatotoxicity in the U.S. 13 , and inadvertent hepatotoxicity is the etiology in half of the cases 14 .
Although most patients experience only mild acute liver injury, such as a transient increase in liver transaminase release resulting in hepatitis and cholestasis, acute liver failure ensues in untreated patients who have ingested large doses. Some acute fulminant hepatic failure patients progress to convulsions, coma, and death. Though the proportion of liver transplants due to ApAP overdose varies depending on the country, it is generally acknowledged that ApAP overdose is a signi cant cause of acute fulminant hepatic failure leading to liver transplantation 15 . Notably, up to 20% of liver transplants in some centers are due to ApAP-associated liver failure 16 .
In response, some countries have implemented restrictions on the sale and availability of ApAP to reduce the risk of accidental and intentional overdoses. Australian regulators recently considered an outright ban on ApAP due to these concerns. However, they ultimately elected to implement restrictions on the maximum number of tablets per package and encourage retailers to limit the number of packages consumers can purchase 17 . In 1998, the U.K. introduced regulations to limit the sale of ApAP in nonpharmacy retail outlets to packs containing a maximum of 16 tablets 18 . In Canada, the risks of ApAP toxicity persisted despite labeling changes implemented in 2009 and 2016 to communicate the risks of ApAP overdose and promote safe use; monthly rates of hospital and intensive care unit admissions for accidental ApAP overdose were unchanged from April 2004 and March 2020 19 .
Because of ApAP's hepatotoxicity, considerable efforts have been devoted to designing safer analgesic and antipyretic analogs 20-23 , including a recent triazole bioisostere 24 . However, non have proven effective beyond the pre-clinical phase. ApAP hepatotoxicity is associated with forming the electrophilic metabolite, N-acetyl-p-benzoquinoneimine (NAPQI) 25,26 , through an oxidative process mediated by CYP2E1 and CYP3A isoforms of CYP450. NAPQI is normally neutralized by a GSH-mediated Phase II and eliminated as a mercapturic acid ( Supplementary Fig. 1,Path A). During overuse, the conjugative Phase II becomes saturated, leading to GSH depletion and accumulation of NAPQI that react with nucleophilic macromolecules, triggering events that result in hepatotoxicity and hepatocellular death ( Supplementary   Fig. 1,Path B), referred to as acetaminophen-induced liver injury (AILI) 27 .

Results
Synthesis rationale of a non-hepatotoxic ApAP analog, SRP-001 To address ApAP's narrow therapeutic index and the clinical need for safer non-opioid pain relievers, we explored ApAP analogs without hepatotoxicity 28 . Our goal was to circumvent toxicity by developing ApAP analogs that connect a saccharin moiety to ApAP's methyl group. To achieve this, we employed an effective synthesis method that involved opening the ring of the heterocyclic moiety, leading to moderately lipophilic compounds, with the R1 and R2 groups being adjustable to in uence lipophilicity.
The lead compound, SRP-001 (Fig. 1d), was chosen due to the absence of in vitro hepatotoxicity and effective in vivo analgesia; the lack of in vitro hepatotoxicity was further validated in vivo.
SRP-001 is devoid of hepatotoxicity due to lack of NAPQI formation and protection of hepatic tight junctions integrity CD-1 mice exposed to toxic doses of ApAP or SRP-001 (600 mg/kg) demonstrate hepatotoxicity, while mice treated with SRP-001 have no hepatotoxicity. Liver sections of ApAP-treated mice displayed centrilobular necrosis and brown nitrotyrosine/diaminobenzidine-positive staining, while SRP-001 lacks nitrotyrosine staining (Fig. 1a). Moreover, SRP-001maintains "chicken wire" tight junctions between hepatocytes, as evidenced by Zonula Occludens (ZO-1) staining (Fig. 1a,b), known to be disrupted by ApAP-hepatotoxicity 29 . Similarly, liver sections from ApAP-treated animals show prominent TUNELpositive apoptotic nuclei, unlike SRP-001-treated mice ( Supplementary Fig. 2b,c). cells were arranged according to expression pro le similarity in a 2-D UMAP scatter plot to show their distribution. The UMAP plot shows that for each sample -Vehicle, CFA_Vehicle, CFA_ApAP, and CFA_SRP-001 -similar cell clusters were retained, and annotated into 10 principal cell types -astrocytes, serotonergic neurons, glutamatergic neurons, GABAergic neurons, mature neurons, tanycytes, endothelial cells, microglia, oligodendrocytes, and oligodendrocyte precursor cells (OPCs) (Figs. 4b-e and 5a). Cells were annotated with differentially expressed marker genes using R package scType described in Methods (Fig. 5a). Feature plots for the top marker gene in each major cluster was made, and UMAP plots for annotated clusters for each group show differences in cell type distribution and quantity (Figs. 4b and 5af). Cell counts for each cell type between treatment groups shows similarities in quanti cation of microglia, oligodendrocyte precursor cells, and oligodendrocytes between Vehicle and CFA_SRP-001 ( Supplementary Fig. 11).
Differential gene expression of neurons and other cell clusters of PAG Differential expression analysis between groups Vehicle vs. CFA_Vehicle, CFA_Vehicle vs. CFA_ApAP, and CFA_Vehicle vs. CFA_SRP-001 was performed using DESeq2, and the top 50 DE genes are shown in heatmaps illustrating that both ApAP and SRP-001 are predicted to regulate similar gene regulatory pathways and that SRP-001 changes the effects based on fold regulation prediction scores ( Supplementary Fig. 12). We also investigated a compendium of known and validated pain-related genes 37,38 and de ned the differential expression of these genes across sample comparisons -CFA_Vehicle vs Vehicle, CFA_ApAP vs CFA_Vehicle and CFA_SRP_001 vs CFA_Vehicle and also across different cell clusters between these samples. Dot plots show that several of these genes are differentially expressed across samples and similar among CFA_ApAP and CFA_SRP-001 treatment groups. Genes were separated based on classi cation as ion channel-, G-protein coupled receptor-, or transcription factor-associated (Supplementary Fig. 13-15).

Gene ontology enrichment analysis
Gene ontology (GO) utilizes the GO knowledgebase to interpret large-scale molecular biology experiments with the endpoint of de ning statistically signi cant similarities or differences between experimental conditions 39 . The knowledgebase provides computational representation of the function of genes and how the genes contribute to a biologic process. Differential expression of genes between Vehicle and CFA_Vehicle treated groups showed enrichment in tau protein binding, molecular carrier activity, peptide binding, GTP binding, and amyloid-beta binding as well as other GO terms ( Supplementary Fig. 20a). The linkage between these terms based on similar genes are shown, and the speci c gene role, in particular, GO terms was visualized ( Supplementary Fig. 20b-c). Differential expression of genes involved in multiple types of gated channel, cation/ion channel, and potassium channel activity was observed from the comparison of CFA_Vehicle and CFA_ApAP ( Supplementary Fig. 21). The comparison between CFA_Vehicle and CFA_SRP-001 de ned differentially expressed genes enriched in GO terms for glutamate receptor activity, glutamate binding, calcium channel activity, and multiple other terms related to ion channel activity like the observed terms from CFA_ApAP ( Supplementary Fig. 22).
Genotoxicity, safety pharmacology, and in vivo non-clinical toxicology To transition SRP-001 into the clinic for human use, a full set of in vivo and in vitro genotoxicity studies were performed to assess the potential genotoxicity of SRP-001. This included a full battery of International Conference on Harmonization (ICH) compliant studies, including in vitro Ames and mammalian chromosomal aberration assays, in vivo mammalian micronucleus and Comet assays indicating that SRP-001 is not genotoxic. Further, SRP-001 has no effects on pulmonary function or cardio-telemetry and is non-cardiotoxic (Table 1 and Online Methods).
To determine the appropriate doses, maximum tolerated dose studies (non-GLP) were conducted prior to the de nitive GLP systemic toxicity studies. SRP-001 was evaluated by a full battery of GLP-compliant toxicity studies. Systemic toxicity was evaluated in Sprague-Dawley rats and Beagle dogs by oral administration over 28 days (Supplementary Fig. 23 and Supplementary Table 2). Importantly, no treatment-related adverse effects were seen during 28 days of daily oral doses of SRP-001 in rats at doses up to 1500 mg/kg/day or in dogs at doses up to 330 mg/kg/day. Single doses of up to 2000 mg/kg in the rat and 1200 mg/kg in the dog of SRP-001 did not result in any mortality, whereas all ApAPtreated mice expired following a 900 mg/kg dose (Online Methods).

Phase 1 trial interim results
A randomized, double-blind, placebo-controlled Phase 1 trial (ClinicalTrials.gov identi er: NCT05484414) is being conducted to assess safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of ascending doses of SRP-001 ( Fig. 3h and Online Methods). The trial is being conducted in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), Guideline for GCP: Consolidated Guidance (E6), attained Institutional Review Board approval, and all applicable regulatory requirements for Phase 1, First in Human clinical trials. Safety measurements and plasma samples to determine PK parameters were collected throughout the study for all subjects and reviewed by the Safety Review Committee.

Discussion
Narcotics for pain management is linked to opioid use disorder (OUD) due to its abuse potential 40 . Drug overdose deaths have risen over the past two decades in the U.S. and, in 2021, accounted for 107,000 deaths 41 . Over 80,000 were from opioids, including highly potent synthetic opioids like fentanyl. Though it is di cult to determine the exact number of opioid overdose deaths that began as prescription opioid users, it is estimated that many individuals who overdosed on opioids initially used prescription opioids for pain management. According to the National Institute on Drug Abuse (NIDA), around 21-29% of patients prescribed opioids for chronic pain misuse them, and about 8-12% develop OUD. Additionally, an estimated 4-6% of those who misuse prescription opioids transition to using heroin 42 . Although ApAP is most used to treat short-term pain, hepatotoxicity is a risk from overuse, and it is the most common cause of fulminant hepatic failure in patients with compromised liver function. And NSAID overuse carries risks of GI bleeding and nephrotoxicity. The high levels of opioid addiction, misuse, and overdose and the toxicity pro les of currently available pain medications underscore the need for safe, effective, non-opioid pain medications.
Owing to ApAP's hepatotoxicity, we characterized SRP-001 from a library 28 of ApAP analogs that lacked hepatotoxicity and exhibited analgesia and antipyresis. SRP-001 is not hepatotoxic because it does not generate NAPQI or disrupt hepatic tight junctions, which are hallmarks of ApAP hepatotoxicity ( Fig. 1 and Supplementary Fig. 2). For the in vivo antinociception studies we used reliable, reproducible animal models and assays of acute and chronic pain used in the preclinical testing of novel and translationally relevant analgesics ( Fig. 2 and Supplementary Figs. 3-7). CFA is a chronic in ammatory pain model, and eVF and Hargreaves are the detection methods to measure mechanical (eVF) and thermal (Hargreaves) nociceptive sensitivity, respectively. Taken together, these pain models suggest that SRP-001 produces e cacious analgesia across various acute and chronic pain conditions, with a larger therapeutic index than ApAP and broad e cacy.
Despite being available since the 1950s, ApAP's analgesia MOA remains to be fully discerned. Increasing evidence, however, strongly supports that AM404 32,33 is fundamental to its analgesia. In the liver, ApAP is converted to p-aminophenol, which, in turn, is converted by FAAH in the brain in the presence of arachidonic acid to make AM404. Current understanding of ApAP's MOA analgesia is through AM404 via the endogenous cannabinoids through CB1 receptors and activation of the TRPV1 channel-receptor signaling in the midbrain PAG 30, 35 . Here, we demonstrate after ip-injection of D 10 -SRP-001 in rat brain and also that SRP-001 produces more AM404 than ApAP in the PAG (Fig. 3) where FAAH/CB1/TRPV1 channel triad co-locates 30 . Beyond this, the central mechanisms for ApAP's analgesia are still unclear.
By examining FAAH gene expression interaction networks, we found similarities in gene modulation related to pain signaling between ApAP and SRP-001 (Figs. 4 and 5 and Supplementary Figs. 9-22). Downregulation of FAAH, CORO2A, and RPL7L1 measured directly from scRNAseq was observed with both ApAP and SRP-001. FAAH is a key enzyme in pain signaling by modulating endocannabinoid levels and lipid-based signaling mediators that regulate physiological processes, including pain perception. The primary function of FAAH is degradation of anandamide (AEA), a main endocannabinoid involved in pain modulation. The role of FAAH in pain signaling has been extensively studied, and it has been found that inhibition of FAAH activity can lead to an increase in AEA levels, reducing pain perception. This process is mediated by the activation of CB1 and CB2 receptors 43 . In addition to AEA, FAAH also degrades other bioactive fatty acid amides, such as oleamide and palmitoylethanolamide, which possess analgesic properties 44 . Directly measured expression from scRNAseq data shows decreased expression of FAAH and genes related to the molecular function of FAAH by SRP-001 and ApAP, indicating that the bioactivity of SRP-001 produces an analgesic effect through FAAH inhibition.
Mechanical nociception gene networks displaying genes that are involved in this biological function also demonstrate the similarities between gene modulatory mechanisms of SRP-001 and ApAP. Activation of vlPAG-located TRPV1 channels, which are expressed on glutamatergic neurons, is required by ApAP to exert analgesic effects via AM404. Activation of these receptors produces analgesic effects through increasing glutamate release to act on mGlu 5 receptors. ApAP has recently been shown to increase glutamate and GABA levels in the presence of an acute pain stimulus in a FAAH-dependent manner 45 . Ion channels present another key target for pain modulation due to their localization in primary sensory neurons. Various receptors and ion channels play a critical role in determining neural excitability; vlPAG pain transmission through descending facilitatory effect can potentially contribute to the development and maintenance of hyperalgesia, which is dependent upon neuron network activity. Previously, calcium and sodium gated ion channels were the focus of pain signaling, but recently, potassium channels have emerged as a potential target for novel analgesics 46 . Potassium channels are necessary to sustain resting membrane potential and repolarizing neurons after an action potential; thus, several potassium channels have been implicated in pain modulation in the PAG area, including the ATP-sensitive potassium (K-ATP) channels, the calcium-activated potassium (KCa) channels, and the two-pore domain potassium (K2P) channels 47 . Concerted activation of proteins, including ion channels, can lead to peripheral sensitization, thus, inhibiton of ion channel activity can reduce neuronal excitability and prevent this peripheral sensitization. Modulation of mechanical nociceptive genes TRPV4, TRPV1, ASICS3, TRPA1, KCNA1, and KCNT1 by SRP-001, provides an early indication of broad ion channel inhibition and potential MOA.
Despite the urgent need for safer and more effective pain medications is widely recognized, particularly considering the opioid epidemic in the U.S., the lack of innovation in this space has left patients with limited options. Backpedaling on 2016 guidelines urging physicians to restrict the use of opioids for moderate-to-severe acute and chronic pain, the Centers for Disease Control and Prevention recently updated its Clinical Practice Guidelines for Prescribing Opioids 48 . The high failure rate of novel pain therapeutics in clinical studies compared to other elds in medicine, the limited availability of investor funding due to this increased risk, and the poor appetite for pain therapeutics among pharmaceutical companies 49 are causes for marginal innovation toward safer and effective pain therapeutics. In order to reduce the developmental risk, these considerations emphasize the signi cance of determining whether a novel pain therapeutic candidate alleviates pain via clinically validated MOA. Thorough pre-clinical safety evaluations, AM404 and transcriptomic studies showcasing a validated, clinically established MOA similar to ApAP, combined with Phase 1 safety data, collectively suggest that advancing the development of SRP-001 could potentially provide a safe and effective pain relief option for acute and chronic pain in humans, characterized by a large therapeutic window. We recently synthesized a library of 2-(benzenesulfonamide)-N-(4-hydroxyphenyl) acetamide analgesics in search of non-hepatotoxic ApAP analogs 28 . From this library, SRP-001 was chemically synthesized using readily available commercial analytical grade reagents of the highest quality, which were purchased and used without further puri cation. Male CD1 mice were fasted overnight for 15 h and dosed with ApAP or SRP-001 or vehicle (0.9% saline) at doses of 600 mg/kg administered via per os (PO) injections with an injection volume of 10 mL/kg body weight. After drug administration, animals were returned to their respective cages and maintained with food and water provided ad libitum for the next 12 h. Animals were then euthanized under 5% iso urane anesthesia after 12 h, and whole blood samples were collected transcardially in sterile microcentrifuge tubes without anti-coagulants. Whole blood samples were stored at room temperature (25 °C) for 30 min, allowing them to coagulate, which was centrifuged at 1,000 g for 5 min at 4 °C to isolate serum samples from whole blood, which were collected, aliquoted, and stored at -80 °C for identi cation of NAPQI, and liver function tests (LFTs).

LC-MS/MS method for NAPQI identi cation and characterization of novel benzoquinoimine
N-acyl-p-benzoquinone imine (NAPQI) was extracted from the serum by adding 3 volumes of ethylacetate to 1 volume of serum and storing them on ice for 30 min, followed by centrifugation at 3000 g for 30 min. Afterward, the supernatant was transferred into mass spectrometry vials and dried under N 2 gas.
The serum was then washed with another 3 volumes of ethyl-acetate, and the supernatant was added back to the same mass spectrometry vial, followed by evaporation under a stream of N 2 gas. The sample was re-suspended with 50 µl of 1: Histology and immunohistochemistry for Nitrotyrosine, hepatic tight junctions, and TUNEL apoptosis assays Male CD1 mice fasted overnight with only access to water ad libitum were dosed with either ApAP, or SRP-001 or vehicle (0.9% saline) (n=5 for each treatment group) via PO injection at concentrations of 600 mg/kg with an injection volume of 10 mL/kg body weight. At 12 h post-injection, mice were deeply anesthetized under 5% iso urane for more than 5 min and formalin-xed via transcardial perfusion with 10% Neutral Buffered Formalin (NBF) after exsanguination using 0.9% NaCl. Liver tissues were extracted and stored in NBF for 24 h post xation, after which they were then transferred to 80% EtOH for storage prior to para n embedding, sectioning, and immunological staining. FFPE blocks were sectioned into 5 µm thick slices on regular frost-free plus slides and dried overnight on a slide warmer. After depara nation, sections were stained with validated antibodies for nitrotyrosine and ZO-1 (tight junctions). For detection of apoptotic nuclei by TUNEL staining, we used Promega's DeadEnd uorometric TUNEL assay, following the manufacturer's suggested protocol. Nitrotyrosine labeling was detected with 3'3'-Diaminobenzidine (DAB) staining, and nitrotyrosine labeled hepatic sections were imaged at 100x magni cation in bright eld using a Nikon Eclipse TS100 microscope with NIS-Elements Excellence vivarium for at least seven days before experimental protocols began. All animals were kept in a 12 h day-night cycle with food and water available ad libitum.

In vivo analgesia models and ED 50 calculation
We explored the antinociceptive properties of SRP-001 usingthe oral nanosuspension of SRP-001. The oral nanosuspension formulation containing 100 mg/mL SRP-001 in preserved aqueous 1% hydroxypropyl cellulose (1% HPC) (Lot# LPI-2021028) was compared to Acetaminophen (ApAP) 100 mg/mL and a vehicle control, in three different in vivo pain animal models -namely, Complete Freund's Adjuvant (CFA) in ammatory, tail ick somatic, and abdominal writhing visceral assays, along with electronic von Frey (eVF) and Hargreaves determination of mechanical and thermal sensitivity.
The investigators were blinded till the completion of all the experiments. To ensure objectivity, investigators were blinded for the acetic acid and tail ick experiments. In the CFA/von Frey and Hargreaves assays, one investigator did the drug pre-treatments and another investigator at another location who was not aware of the treatments conducted the behavioral testing. Moreover, these studies used the eVF device that electronically registers the grams of force necessary to elicit paw withdrawal, eliminating bias that confounded older versions of this test in which an investigator used a series of individual von Frey micro laments to determine paw withdrawal thresholds. Six separate experimenters were involved in the data acquisition for behavioral testing and another separate investigator performed all the statistical analyses using GraphPad Prism Version 9.1.2. Statistical signi cance was determined by p<0.05; one-way ANOVA followed by Sidak's multiple comparisons post hoc test. Mixed-gender experiments were powered to test for gender effects, and different ages of rodents were also included for the experiments -young (2 months) and aged (20 months) to tease out whether there are any effects of age-related changes in nociceptive sensitivity under naïve, and in ammatory pre-clinical rodent pain models. For the aged rats and mice, we could only obtain male Sprague-Dawley rats and male C57BL/6 mice of 20 months old from all commercial animal vendors and from the National Institute of Aging (NIA). So, only these rodents were used for the older animal experimental cohort. Old female rats and mice were not available at the time when these experiments were conducted. For ED 50 calculation, dose response curves were calculated with regression analysis and log transformation using GraphPad Prism 9.1.2.

CFA/von Frey with electronic detection (eVF)
First, we used male Sprague-Dawley rats and the CFA in ammatory pain assay with electronic von Frey detection to assess antinociceptive/anti-hyperalgesic e cacy of SRP-001 compared to ApAP and vehicle control. In this model of mechanonociception, one hind paw at a time is stimulated with an electronic von Frey (eVF) lament (noxious source) until the animal retracts the paw from the mechanical stimulus. von Frey tests were conducted in a dedicated room at the LSUHSC New Orleans Neuroscience Center of Excellence vivarium. Rats were acclimated to their environment for 2 days for 30 min/day prior to testing.
To obtain eVF pressure recordings, each animal was placed in an individual plastic observation compartment on a perforated metallic grid platform, which provided access to the plantar surface of the hind paws. After acclimation to the environment for 30 min, mechanical hypersensitivity was assessed by stimulating the mid-plantar area of each hind paw with a rigid tip von Frey lament attached to the eVF meter (Ugo Basile 38450) until animals withdrew the paw from the lament. The withdrawal threshold was de ned as the average force/pressure (g) required for the rat to withdraw the stimulated paw. A brisk withdrawal of the paw (often followed by a sustained retraction and/or licking) was considered a positive response, but paw withdrawals due to locomotion or weight shifting were not counted. The von Frey studies were conducted with an eVF device that electronically registers the grams of force necessary to elicit paw withdrawal in order to eliminate bias that confounded previous older versions of this test.
Baseline withdrawal thresholds of both the right and left paws were recorded, and animals were assigned to different treatment groups so that each group had approximately equal withdrawal threshold averages in both paws. After baseline testing, treated animals received subcutaneous plantar injection (150μl) of 50% CFA into the left hind paw, while control animals received a plantar injection (150µl) of 0.9% NaCl into their left hind paw. CFA induces in ammation, resulting in a left hind paw that is hypersensitive to mechanical stimulus, while the right hind paw serves as a within-subject baseline for each animal. On the day of testing, each animal received the drugs -ApAP or SRP-001 via PO administration based on their assigned treatment groups. Injections at concentrations of 32 mg/kg and 100 mg/kg were tested in a cumulative dose-response manner and given at an interval of 60 min, and paw withdrawal threshold readings were measured using eVF as described above.
Young Male Rats. In this cohort, n = 40 male Sprague-Dawley rats (2 months) were used, and two different doses of SRP-001 oral nanosuspensionand ApAP (32 and 100 mg/kg) were compared to a vehicle control.
Young Female Rats. In this cohort, n = 40 female Sprague-Dawley rats (2 months) were used, and two different doses of SRP-001 oral nanosuspensionand ApAP -32 and 100 mg/kg were compared to a vehicle control.
Aged Male Rats. In this cohort, n = 20 male Sprague-Dawley rats (20 months) and two different doses of SRP-001 oral nanosuspensionand ApAP -32 and 100 mg/kg were compared to a vehicle control.
We used cold stimulation tail ick somatic pain assay testing 2 different doses of ApAP or SRP-001 at 32 m/kg or 100 mg/kg to measure increased tail withdrawal time (latency) in CD1 (young male and female) or C57BL/6 (aged male) mice to cold stimulation of the tail tip. For the tail-ick assays, we used n = 70 aged male mice, n = 90 young male mice, and n = 120 young female mice. Tail ick assay experiments were conducted on an open bench in a dedicated room at the LSUHSC New Orleans Neuroscience Center of Excellence vivarium. Mice were allowed to acclimate to the laboratory environment for 1 h prior to testing. To restrain the mice for the test, disposable plastic 50 mL screw-capped conical centrifuge tubes were cut at the tip to create a 0.5 cm opening to allow the mice to breathe freely. Another 0.5 cm opening was cut into the cap to allow access of the tail to the water bath. A 500 mL glass beaker was lled with 450 mL of ice-cold distilled water maintained at 4°C with the addition of ice and determined with a glass thermometer. Mice were held over the opening of the water bath, and their tails submerged approximately halfway into the water. The nociceptive threshold was taken as the latency until the mice icked their tail tip or removed the tail. The time from immersion to the attempted tail tip removal was measured to 1/10 th of a sec with a digital laboratory timer. To minimize damage to the tail, a 60-sec cut-off was utilized. After baseline measurements, mice were injected with ApAP or SRP-001 via PO administration based on their assigned treatment groups. Injections were at concentrations of 32 mg/kg and 100 mg/kg. 30 min post-drug administration tail-ick/withdrawal latency was measured.
Abdominal writhing assay In this model of visceral pain, abdominal contraction (writhing) in which contraction of the abdominal muscles and stretching of the hind limbs is induced in mice in response to an ip injection of 0.4% acetic acid at a dose of 10 mL/kg 25 min after drug administration. The number of writhes is counted for 10 min beginning 5 min after acetic acid injection 51 . All animals were fasted overnight (15 h) prior to testing, and the compounds were administered via PO injection to animals belonging to the treatment groups -ApAP or SRP-001 -and tested at doses of 32 mg/kg and 100 mg/kg, respectively. CD1 (young male and female) or C57BL/6 (aged male) mice were used. For the abdominal writhing assays, we used n = 35 aged male mice, n = 70 young male mice, and n = 70 young female mice.

CFA-induced in ammatory pain/Hargreaves thermal sensitivity antinociception assay
First, we used male Sprague-Dawley rats and the CFA in ammatory pain assay with eVF detection to assess antinociception/anti-hyperalgesic e cacy of SRP-001 compared to ApAP and vehicle control. In these same cohorts of animals, we simultaneously measured their thermal nociception to hyperalgesia with the Hargreaves test using the plantar test apparatus (Ugo Basile 37570). In this experimental setup, the rodents are placed in plastic cages on top of a glass surface, and their hind paws are subjected to an infrared heat stimulus. One hind paw at a time is stimulated with the light source (noxious stimuli) until the animal retracts the paw from the glass surface because of the stimulus. Baseline withdrawal thresholds (latency) of both the right and left paws were recorded, and animals were assigned to different treatment groups so that each group had approximately equal withdrawal threshold averages in both paws. After baseline testing, treated animals received subcutaneous plantar injection (150μl) of 50% CFA into the left hind paw, while control animals received a plantar injection (150µl) of 0.9% NaCl into their left hind paw. CFA induces in ammation, resulting in a left hind paw that is hypersensitive to the thermal stimulus, while the right hind paw serves as a within-subject baseline for each animal. To obtain Hargreaves withdrawal latency recordings, each animal was placed in an individual plastic observation compartment on a glass platform, which provided access to the plantar surface of the hind paws from underneath. After acclimation to the environment for 30 min, thermal hypersensitivity was assessed by stimulating the mid-plantar area of each hind paw with a bright light source attached to the meter (Ugo Basile 37570) until animals withdrew the paw from the surface. The withdrawal threshold was de ned as the average time (s) required for the rat to withdraw the stimulated paw. A brisk withdrawal of the paw (often followed by a sustained retraction and/or licking) was considered a positive response, but paw withdrawals due to locomotion or weight shifting were not counted. The Hargreaves studies were conducted with the plantar stimulation device that automatically registers the time (s) necessary to elicit paw withdrawal in order to eliminate bias in determining paw withdrawal thresholds. A cutoff of 20 s is pre-programmed so that the light source shuts off at that maximal period so as to not induce any burns on the plantar surface of the rodents. The Hargreaves test permits measurement of ipsilateral and contralateral heat thresholds, allowing each animal to serve as its own internal control in unilateral pain models. In addition, the Hargreaves test enables quanti cation of heat thresholds in unrestrained animals, reducing the likelihood of stress-induced responses.
Young Male Rats. In this cohort, n = 40 male Sprague-Dawley rats (2 months) were used, and two different doses of SRP-001 oral nanosuspensionand ApAP -32 and 100 mg/kg were compared to a vehicle control.
Young Female Rats. In this cohort, n = 40 female Sprague-Dawley rats (2 months) were used, and two different doses of SRP-001 oral nanosuspensionand ApAP -32 and 100 mg/kg were compared to a vehicle control.
Aged Male Rats. In this cohort, n = 20 male Sprague-Dawley rats (20 months) were used, and two different doses of SRP-001 oral nanosuspensionand ApAP -32 and 100 mg/kg were compared to a vehicle control.

In vivo antipyresis assays
Antipyresis experiments were carried out with n = 60 CD-1 male mice weighing between 45-50g.All mice were kept in a 12-h day/12-h night cycle with free access to food and water ad libitum. After habituating and acclimating the mice for a week, each mouse was brie y anesthetized for 1 min with 1% iso urane and implanted subcutaneously a transmitter probe that records core body temperature -Implantable Programmable Temperature Transponder (IPTT-300) by Bio Medic Data Systems (BMDS), DE, USA. The mice were allowed to recover for at least one week from the surgery before any recordings were made and observed in their home cages prior to pyrogen challenge for inducing fever. Temperatures were recorded using the BMDS DAS-8027-IUS data reading system.

Pyrogen (LPS) injections
All mice were fasted overnight (15 h) with free access to water ad libitum prior to the pyrogen challenge. Drug injection/concentration: All drugs and saline were administered by oral gavage, PO injection. Each injection volume was ~0.3-0.4mL per mouse. The dose for ApAP and the novel compound, SRP-001, used for this trial was 75mg/kg. We chose this dose to record antipyretic effect in a low dose that doesn't cause hypothermia in mice. Febrile mice were grouped into different control and experimental groups as follows: n=10 for Normal Control (Baseline) n=20 for Positive Control (APAP) and n=20 for SRP-001 Normal Control (Baseline) -0.9% Saline (ip) and 0.9% saline (po) Positive Control (ApAP) -LPS (100 μg /kg) (ip) and APAP (75mg/kg) (po) SRP-001 -LPS (100 μg /kg) (ip) and SRP-001 (75mg/kg) (po) Baker yeast-induced hyperthermia (15% yeast, 0.1mL/kg) (MilliporeSigma, Billerica, MA, USA) was used to induce fever. All animals used in this assay were male CD1 mice, which were fasted overnight (15h) before the start of the assay.
Mice received either yeast in 0.9% saline (sterile) (vehicle) or just 0.9% saline as an ip injection. Mice were returned back to their home cages post-injection and observed, and core body temperatures recorded at 10% DMSO: 80% PEG400) at 10 mL/kg. Following dosing, each animal was returned to its designated plethysmograph chamber, and the respiratory parameters were recorded at 30 min (± 5 min), 2, 4, and 6 h (±15 min). Animals were allowed to stabilize in the plethysmograph chamber for at least 5 min before each reading was taken. The following parameters were acquired, recorded, and analyzed using typhimurium and at the tryptophan locus of E. coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

2) In Vitro Mammalian Chromosomal Aberration Assay in Chinese Hamster Ovary Cells.
In the preliminary toxicity assay, the doses tested ranged from 0.0405 to 405 µg/mL (1 mM), which was the limit dose for this assay. Cytotoxicity (≥ 45% reduction in cell growth index relative to the vehicle control) was not observed at any dose in the non-activated and S9-activated 4-h exposure groups.
Cytotoxicity was observed at doses ≥ 122 µg/mL in the non-activated 20-h exposure group. Based on these results, the doses chosen for the chromosome aberration assay ranged from 75 to 405 µg/mL for the non-activated and S9-activated 4-h exposure groups and from 5 to 125 µg/mL for the non-activated 20-h exposure group.
In the chromosome aberration assay, cytotoxicity (≥ 45% reduction in cell growth index relative to the 3) Clastogenicity and rat micronucleus; in vivo study: SRP-001 is deemed negative (non-clastogenic).
Male rats were dosed at 500, 1,000, and 2,000 mg/kg/d once per day on two consecutive days at 10 mL/kg via oral gavage using 10% labrasol, 10% DMSO, and 80% PEG400 as the vehicle. Approximately 48 h after the second dose administration, peripheral blood was collected for ow cytometric analysis of micronuclei. There was no signi cant increase in the incidence of micronuclei in the test article-dosed animals compared to the concurrent vehicle control. Liver. No statistically signi cant increases in % tail DNA were observed in SRP-001-treated groups. The group mean % tail DNA for the vehicle control was within the 95% control limit of the study historical data range, with one animal (#160) exceeding the individual animal 95% control limit but within the minimum/maximum range. Additionally, three animals (#173, 174, and 175) in the mid-dose (1000 mg/kg/dose) group likewise exceeded the individual animal 95% control limit but were within the minimum/maximum range. The group mean % tail DNA for the positive control was signi cantly increased when compared to the concurrent group mean % tail DNA for the vehicle control and was compatible with the positive control database for both the study and individual animals.
Glandular Stomach. No statistically signi cant increases in % tail DNA were observed in the SRP-001treated groups. The group mean % tail DNA for the vehicle control was within the 95% control limit of the study historical data range, with one animal (#158) exceeding both the individual animal 95% control limit and minimum/maximum ranges. One animal (#171) in the mid-dose (1000 mg/kg/dose) group and one animal (#180) in the high-dose (2000 mg/kg/dose) group exceeded just the 95% control limit range but were within the minimum/maximum range. One animal (#170) in the mid-dose (1000 mg/kg/dose) group and one animal (#179) in the high-dose (2000 mg/kg/dose) group exceeded both the individual animal 95% control limit and minimum/maximum ranges. The group mean % tail DNA for the positive control was signi cantly increased when compared to the concurrent group mean % tail DNA for the vehicle control and was compatible with the positive control database for both the study and individual animals. All valid assay criteria were met, and SRP-001 was determined to be negative (non-DNA damaging) in the Comet assay.

Non-GLP and GLP toxicology 1) Dose-Range-Finding Oral Toxicity Study with SRP-001 in Sprague-Dawley Rats (non-GLP)
As part of a two-phase study, the maximum tolerated dose (MTD) of SRP-001 when administered via gavage once to Sprague-Dawley Rats using an ascending/descending dose design. A total of 12 male Sprague-Dawley rats (3/dose group), approximately 8-9 weeks old and weighing 245-307 g, were administered single doses of 500, 1000, 1500, or 2000 mg/kg SRP-001. Mortality/morbidity was performed twice daily and once prior to scheduled sacri ce. Clinical observations were evaluated prior to each dose administration and approximately 1-3 h post-dose, once daily on non-dosing days and additionally as needed. Body weights were recorded for animals in Groups 1-4 prior to randomization/selection, each dose administration, and scheduled sacri ce on day 3. Food consumption was recorded on day 3. Nearly all the animals appeared normal following dosing, and no test articlerelated effects on body weight or food consumption were observed. All animals appeared unremarkable at gross necropsy. Based on the results of this study, doses for the second phase were selected.

2) A Single Oral Dose Toxicity and Pharmacokinetic Study of SRP-001 in Beagle Dogs (non-GLP)
SRP-001 was prepared into capsules for oral administration. Three experimentally non-naïve Beagle dogs, at least 7 months old and weighing 7.7 to 10 kg prior to treatment initiation, were administered a single dose of 300 mg/kg SRP-001. On the day of dosing, animals were observed prior to dose administration, approximately 1-3 h post-dose, and once on day 2. Body weights were recorded for all animals prior to dose administration for the preparation of the capsules. Blood for bioanalytical evaluation was collected at selected timepoints on day 1. Following the completion of the study, surviving animals were not euthanized. They were returned to the Calvert colony for an appropriate washout period before possible use in a subsequent study. The dose was well tolerated, and all animals appeared normal following dosing. Plasma samples were collected at seven different time points post-dose. Bioanalytical analysis revealed poor bioavailability was achieved following dosing. Capsule dosing, while well tolerated in dogs at 300 mg/kg, was not considered suitable for future studies.

3) A Dose-Range-Finding and 7-day Repeat-Dose Oral Toxicity Study of SRP-001 in Beagle dogs (Non-GLP)
Two beagle dogs (1/sex) were administered 30, 100, 250, or 500 mg/kg SRP-001. Animals were observed for clinical signs of toxicity or effect prior to dose administration and approximately 1-3 h post-dose, additionally as needed, and daily on non-dosing days. After a period of at least 2 d (minimum of 44 h) following the previous dose of test article, additional dose levels of SRP-001, as indicated above, were administered by oral gavage to the same 2 dogs (1 male and 1 female). Each dose was based on the dog's most recent body weight. Mortality/morbidity was observed twice daily and once prior to scheduled sacri ce. Body weights were recorded prior to randomization/selection, prior to each dose administration, and prior to scheduled sacri ce. At scheduled sacri ce, a gross necropsy was performed, and the tissues were appropriately discarded. Loose feces and emesis were observed following most doses; however, no dose-dependent trend was observed. No test article-related effects on body weight or food consumption were observed. At gross necropsy, Phase I animals had some red discoloration in the GI tract, as observed in the duodenum, jejunum, ileum, and rectum. Doses for Phase II of the study were set based on Phase I results.

4) A Dose-Range-Finding Oral Toxicity Study with SRP-001 in Sprague-Dawley Rats (Non-GLP)
As part of a two-phase study, repeat-dose toxicity and toxicokinetics of SRP-001, when administered via oral gavage once daily to Sprague-Dawley rats for 5 d, was done. The doses selected for Phase II were based on the results of Phase I. SRP-001 was administered to 20 naïve rats (5/group in Groups 5-8) once daily for 5 d via oral gavage for toxicology evaluation and once to 18 naïve rats (6/group in Groups 9-11) for toxicokinetic evaluation.
Mortality/morbidity was performed twice daily and once prior to sacri ce. Clinical observations were evaluated prior to each dose administration and approximately 1-3 h post-dose for animals in Groups 5-8.
Body weights were recorded prior to randomization/selection, prior to dose administration on day 1 for animals in Groups 5-11, and prior to dose administration on day 5 for Groups 5-8. Food consumption was recorded for animals from Groups 5-8 on day 5. Blood for evaluation of hematology, coagulation and clinical chemistry and urine for urinalysis was collected from animals in Groups 5-8 on day 6. All animals in Groups 5-8 were sacri ced on day 5. Selected tissues were harvested at necropsy, selected organs weighed, and selected tissues preserved. Blood for toxicokinetic evaluation was collected from animals in Groups 9-11 at selected timepoints on day 1.
Clinical signs included soft feces, brown staining of the anogenital area, and a few instances of red staining of the muzzle. These signs were observed in all groups and are likely related to the vehicle used.
Test article-related increases in absolute and relative liver weights and increased cholesterol levels were observed. Dark discoloration of the liver was observed in the majority of control animals and all experimental animals at gross necropsy. No SRP-001-related differences in body weight, food consumption, hematology, or coagulation parameters were observed. AUC and Cmax levels increased in a dose-dependent manner. Half-lives were 6.19 and 6.86 h at mid and high doses, respectively. Tmax ranged from 3 h at the low dose to 4.5 h at mid and high doses. Based on these observations, SRP-001 was considered to be tolerated at 1200 mg/kg when administered once daily for 5 consecutive days.

5) A 28-day Oral Toxicity Study of SRP-001 in Sprague-Dawley Rats with 14-day Recovery (GLP study)
We next set out to determine the toxicity and toxicokinetics of SRP-001 when administered via oral gavage once daily to Sprague-Dawley Rats once daily for 28 d. The study also assessed the reversibility of any toxicity observed with a 14-d recovery period. One hundred thirty-six experimentally naïve Sprague-Dawley rats (68 males and 68 females), 8 weeks old and weighing 167-295 g for males and females at the outset of the study, were assigned to toxicology treatment groups (Groups 1-4) or toxicokinetic groups (Groups 5-7). For toxicology groups, 10 animals/sex/dose were administered 0, 300, 900, or 1500 mg/kg/d SRP-001. An additional 5 animals/sex/dose in Groups 1 and 4 were targeted for a 14-d recovery period and euthanized on day 43. For toxicokinetic groups, 6 animals/sex/dose were administered 300, 900, or 1500 mg/kg/d SRP-001. Mortality/morbidity was performed twice daily and once prior to scheduled sacri ce. Animals in Groups 1-4 were observed prior to each dose administration and 1-3 h post-dose. Animals in Groups 1-4 were also observed once daily on non-dosing days and once prior to scheduled sacri ce. Body weights were recorded prior to randomization/selection, prior to dose administration on day 1, and weekly thereafter. Food consumption was determined weekly for animals in Groups 1-4. Ophthalmology examinations were performed before treatment initiation and during the last week of dosing for animals in Groups 1-4. The functional observational battery was assessed for 5 animals/sex/group in Groups 1-4 once prior to dosing and at 1-2 h following dose administration on day 1. Blood for evaluation of hematology, coagulation, and clinical chemistry and urine for urinalysis was collected from animals in Groups 1-4 on day 29 or day 43. Blood for toxicokinetic evaluation was collected from animals in Groups 5-7 at selected timepoints on days 1 and 28. All surviving animals in Groups 1-4 were sacri ced on day 29 or day 43. Selected tissues were harvested at necropsy, selected organs weighed, and selected tissues evaluated microscopically.
Following dosing, the reversibility of any toxicity observed was assessed with a 14-d recovery period of animals receiving 0 or 1500 mg/kg of SRP-001. SRP-001-related clinical signs included red staining around the forepaw, forelimbs, mouth, muzzle, and nose and brown-staining in the anogenital area. To some extent, these clinical observations appeared to be vehicle-related; however, they were observed at an increased frequency and duration correlating with increasing dose levels. The majority of clinical signs were observed during the rst 3 weeks of dosing, and by day 28, the majority of the animals appeared normal. Hematology and serum chemistry analysis on day 29 identi ed non-adverse SRP-001-related increases in globulin, albumin, and total protein and decreases in aspartate aminotransferase, alkaline phosphatase, and mean corpuscular hemoglobin concentration. These differences remained within or close to the historically normal ranges. Statistically signi cant differences in total protein, globulin, and mean corpuscular hemoglobin remained present in males after the recovery period, although at a smaller magnitude. SRP-001-related microscopic ndings were limited to the liver and included minimal to mild centrilobular hepatocellular hypertrophy in both sexes at ≥300 mg/kg/d. This nding correlated with statistically signi cant increases in liver weights in both sexes. Hepatocellular hypertrophy was considered to be an adaptive response and non-adverse due to limited severity, lack of histological evidence suggestive of structural damage (such as a concurrent dose-dependent increase in hepatocellular necrosis or in ammation), and lack of dose-dependent and biologically signi cant increases in clinical chemistry parameters suggestive of hepatobiliary damage.
There were no SRP-001-related effects on body weight, food consumption, functional observation battery, ophthalmological ndings, coagulation parameters, or urinalysis parameters. Exposure to SRP-001 following oral gavage dosing at 300, 900, and 1500 mg/kg SRP-001 was dose-dependent, increasing with escalating doses following a single dose. Exposure to SRP-001 following 28 consecutive days of oral gavage doses at 300, 900, and 1500 mg/kg/d SRP-001 was dose-dependent but did not increase as much as seen on day 1. Based on the parameters observed, the no-adverse effect level (NOAEL) of SRP-001, when administered once daily for 28 d to Sprague-Dawley rats, was 1500 mg/kg.
Cardiotoxicity is also absent with SRP-001. This stems from no cardiovascular effects in 28-day GLP studies in both beagle dogs or rats and in a telemetered safety pharmacology study in dogs. It did not result in a relevant signal in the in vitro hERG assay in human embryonic kidney cells at dosages where acetaminophen causes signi cant changes in these markers is absent with SRP-001. SRP-001 was a weak blocker of hERG current with the highest concentration tested (100μM) blocking by 21.4 ± 3.9% (Supplementary Fig. 1d) Body weights were recorded for all animals prior to the rst dose administration on days 1 and 3, and food consumption was recorded daily. Blood for bioanalytical evaluation was collected at selected timepoints on day 1. Following the completion of the study, surviving animals were not euthanized and were returned to the Calvert colony.
Nearly all animals had emesis following each dose administration. Other clinical signs observed included loose or watery feces and salivation. Nearly all animals appeared normal prior to receiving their next dose. Body weight loss was also observed; however, no animal lost more than 4% of their body weight.
Body weight loss correlated with decreased food consumption on day 3. Bioanalytical analysis of plasma con rmed SRP-001 exposure in all animals. Overall, SRP-001 was tolerated when administered 3 times daily for 2 d at 900 mg/kg/d, and plasma exposure was observed.

7) A Dose-Range-Finding and 7-day Repeat-Dose Oral Toxicity Study of SRP-001 in Beagle dogs (Non-GLP)
In Phase II of this study, 0, 30, 100, or 300 mg/kg/d of SRP-001 was administered once daily for seven consecutive days to 1 Beagle dog/sex/dose. Mortality/morbidity was observed twice daily and once prior to scheduled sacri ce. Clinical observations were evaluated prior to each dose administration and at approximately 1-3 h post-dose and once prior to scheduled sacri ce on day 8. Body weights were recorded on days 1 and 7, and a fasted body weight was recorded prior to scheduled sacri ce on day 8.
Food consumption was recorded daily. Blood for evaluation of hematology, coagulation, and clinical chemistry parameters and urine for urinalysis was collected from all animals prior to treatment initiation and from all Phase II animals prior to scheduled sacri ce on day 8. Blood for toxicokinetic evaluation was collected at selected time points on days 1 and 7. Selected tissues were harvested and weighed at necropsy. No microscopic evaluations were performed. Clinical signs observed included loose or soft feces and emesis. While soft or loose feces were observed at similar frequencies in all animals, emesis was observed at a higher incidence and greater volume in animals that received higher dose levels. The clinical observations corroborated with ndings of red discoloration in the duodenum of the control group female, the jejunum of the low-dose male, and the colon of both high-dose dogs. No SRP-001-related differences in body weight, food consumption, hematology, clinical chemistry, or coagulation parameters were observed.
AUC and Cmax levels increased in a dose-dependent manner, with day 1 levels higher than those from day 7. The reason for this is unclear; however, it may be due to a low sample size (n=2 per dose group).
Half-lives ranged from approximately 1 to 3.5 h and increased with dose and time. Tmax ranged from 0.5 to 1.5 h. Based on these observations, SRP-001 was considered to be tolerated at 300 mg/kg when administered once daily for 7 consecutive days. In-life data from the initial start of the study using once daily dosing were not included in the report but are maintained in the study le. Following the restart of the study, the study was conducted as follows.

8) A 28-day Oral Toxicity Study of SRP-001 in Beagle
Mortality/morbidity was performed twice daily and once prior to the scheduled sacri ce. Animals were observed prior to each dose administration and 1-3 h after each dose administration. Animals were also observed once daily on non-dosing days and once prior to scheduled sacri ce. Body weights were recorded prior to randomization/selection, prior to the rst dose administration on day 1 and weekly thereafter. Food consumption was determined daily. Ophthalmology examinations and electrocardiograms were performed before treatment initiation and during the last week of dosing. Blood for evaluation of hematology, coagulation and clinical chemistry and urine for urinalysis was collected prior to treatment initiation and on days 29 and 43. Blood for toxicokinetic evaluation was collected at selected timepoints on days 1 and 28. All surviving animals were sacri ced on day 29 or day 43. Selected tissues were harvested at necropsy, selected organs weighed, and selected tissues evaluated microscopically. SRP-001-related clinical signs included emesis, loose feces, and salivation. While emesis and loose feces were observed in all dose groups, including the control, they were observed at a higher frequency and severity with increasing dose levels.
Assessment of hematology parameters identi ed SRP-001-related decreases in red blood cells, hemoglobin, hematocrit, and mean corpuscular hemoglobin concentration and increases in mean corpuscular volume and absolute and relative reticulocytes. These changes correlated with increased erythroid cellularity of the bone marrow and extramedullary hematopoiesis of the spleen and liver, indicative of regenerative anemia. Due to the limited magnitude of the changes in hematology parameters and the corresponding changes in reticulocytes, this nding was not considered adverse.
Assessment of clinical chemistry parameters indicated SRP-001-related increases in triglycerides and alkaline phosphatase and decreases in cholesterol. Increases in alkaline phosphatase correlated with bile accumulation in the liver of Group-4 males. This nding was indicative of the biliary system starting to be overwhelmed and was considered adverse. Other microscopic ndings of the liver included Kupffer cell pigment, Kupffer cell erythrophagocytosis, and Kupffer cell hypertrophy/hyperplasia. The Kupffer cell changes were thought to be, at least in part, due to phagocytosis of erythrocytes resulting in the accumulation of cytoplasmic pigment and increased demand for Kupffer cells resulting in hypertrophy/hyperplasia. These microscopic changes correlated with increased liver organ weights.
Microscopic evaluation also identi ed thyroid gland follicular cell hypertrophy.
No SRP-001-related changes were observed in body weights, food consumption, ophthalmology examination, electrocardiology examination, coagulation parameters, or urinalysis parameters. Analysis of dogs following a 14-d recovery period indicated that nearly all SRP-001-related changes observed on day 29 were reversible. Only microscopic changes in Kupffer cell pigment and erythrophagocytosis and minimal increased erythroid cellularity of the bone marrow were still present in Group-4 animals following the recovery period. Exposure to SRP-001 was dose-dependent and generally dose-proportional to greater than dose-proportional from 165 to 330 mg/kg/d and less than dose-proportional from 330 to 495 mg/kg/d. Exposure to SRP-001 following 28 consecutive days of oral dosing was 2 to 5 times less than exposure observed on day 1. Based on the clinical signs, microscopic changes, and clinical pathology changes, the no-adverse-effect level for SRP-001 was 330 mg/kg/d when given T.I.D. for 28 d.
Oral nanosuspension formulation for human use and determination of the optimal formulation stability The drug therapeutic candidate is manufactured in a two-step process. First, SRP-001 is jet milled. Second, the milled API is dispersed in a solution comprising 1% hydroxypropyl cellulose (1% HPC) and sterile water for injection and subjected to wet milling. The target particle size is approximately D 90 0.15 μm. Hence, using nanomilling and jet-milling techniques, we identi ed the optimal solvent in which to dissolve to develop SPR-001 as an oral nanoparticle suspension containing 100 mg/mL SRP-001 in preserved aqueous 1% HPC (Supplementary Fig. 24). An oral nanoparticle suspension containing 100 mg/mL SRP-001 in aqueous 1% HPC preserved with 0.1% sodium benzoate was manufactured for use in nonclinical studies as Lot LPI2021028 and stored at 5°C ± 3°C. All speci cations were met at time 0, 1 month, and 3 months at 5°C and 25°C/60% RH (relative humidity). The same formulation as above was manufactured under cGMP for use in clinical studies as Lot LPI2021031 and stored at 5°C ± 3°C. All speci cations were met. This lot has been placed on stability at 5°C, 25°C/60%RH, and 40°C/75%RH.
Finally, an appearance-matched placebo consisting of 2% microcrystalline cellulose in aqueous 1% HPC preserved with 0.1% sodium benzoate and 0.05% EDTA disodium dihydrate was manufactured under cGMP for use in clinical studies. The above oral suspensions were manufactured by Latitude Pharmaceuticals Inc. (San Diego, CA, USA). Furthermore, the active pharmaceutical ingredient (API, SRP-001) manufacturer, Olon Ricerca Biosciences, demonstrated that the API is stable for at least 9 months at 25°C/60%RH and 6 months at 40°C/75%RH.

Human studies
A two-part randomized, double-blind, placebo-controlled Phase 1 trial was conducted to assess the safety,  Table. Females must not be pregnant or breastfeeding. The subjects must be capable of understanding the Informed Consent Form and agreeing to comply with the requirements of the study during its duration. The subjects are to be in good health as determined by medical history, physical examination, clinical laboratory test results, and 12-lead ECG. Females must be of non-childbearing potential (de ned as surgically sterile (i.e., had a bilateral tubal ligation, bilateral salpingectomy, hysterectomy, or bilateral oophorectomy) or post-menopausal for at least 1 year before the rst dose of study medication) or agree to use an acceptable form of birth control from screening until 31 days after last SRP-001 administration. Male subjects must either be surgically sterile (vasectomy at least 3 months prior to rst dose) or agree to the use of a birth control method such as a condom with spermicide from screening until 91 days after last SRP-001 administration. All subjects must also be willing and able to remain in the research center for the entire duration of the con nement period and return for the outpatient visits; and, have vital signs (measured after the subject has been in a supine position for a minimum of 5 min) at screening within the following ranges: heart rate: 40-100 bpm; systolic blood pressure (BP): 90-145 mmHg; diastolic BP: 50-95 mmHg. Out-of-range vital signs may be repeated once.
Exclusion criteria: NHVs were excluded from study participation for any of the following. A history or presence of clinically signi cant cardiovascular, pulmonary, hepatic, renal, hematologic, GI, endocrine, immunologic, dermatologic, neurologic, oncologic, or psychiatric disease or any other condition that, in the opinion of the Investigator, would jeopardize the safety of the subject or the validity of the study results (a clinically signi cant illness within 30 days preceding the screening visit). If he or she had been on a signi cantly abnormal diet during the 4 weeks preceding the rst dose of study medication. Subjects who have received any investigational product in a clinical research study within 5 half-lives or within 30 days prior to rst dose. However, in no event shall the time between the last receipt of investigational product and the rst dose be less than 30 days. Any NHVs who have previously been administered SRP-001 in this study. Subjects who are taking, or have taken, any prescribed drug (other than hormone replacement therapy/hormonal contraception) in the 14 days before study medication, or OTC drug or herbal remedies in the 72 h before study medication. COVID-19 vaccines are accepted concomitant medications. NHVs were also excluded if they had been treated with any known drugs that are moderate or strong inhibitors/inducers of cytochrome P450 (CYP) enzymes (e.g., barbiturates, phenothiazines, cimetidine, carbamazepine) within 30 days before the rst dose of study medication, and that, in the Investigator's judgment, may impact subject safety or the validity of the study results; if they had a history of hypersensitivity (has developed an allergic reaction) to acetaminophen or similar chemical entities; or presence or history of clinically signi cant allergy requiring treatment, as judged by the Investigator. The NHV's hemoglobin concentration and hematocrit should be within 5% of normal before participating in the study. It is recommended that blood/plasma donations not be made for at least 30 days after discharge from the study. Current smokers and those who have smoked within the last 12 months were also excluded, as anyone with a con rmed positive urine cotinine test at screening or admission; current users of e-cigarettes and nicotine replacement products and those who have used these products within the last 12 months; consumption of beverages or foods that contain alcohol, grapefruit, poppy seeds, broccoli, Brussels sprouts, pomegranate, star fruit, char-grilled meat, or caffeine/xanthine from 48 h before the rst dose of study medication until discharge from the study. NHVs were instructed not to consume any of the above products. Also excluded is an NHV with a con rmed positive alcohol urine test at screening or admission; a female with a positive pregnancy test result; or a positive urine screen for drugs of abuse (amphetamines, barbiturates, benzodiazepines, cocaine, cannabinoids, or opiates); a positive test for hepatitis B surface antigen (HBsAg), hepatitis C antibody (HCV Ab), or human immunode ciency virus (HIV) at screening or has been previously treated for hepatitis B, hepatitis C, or HIV infection. Anyone with the presence of active infection, mucositis, cold sores, aphthous ulcers, vesicles, viral lesions, local irritation/in ammation, or periodontal disease of the oral cavity was also excluded, as well as NHVs with a known glucose-6-phosphate-dehydrogenase (G6PD) de ciency.
Subject randomization scheme: NHVs were assigned a three-digit screening number after informed consent was obtained (e.g., 001, 002, 003, etc.). Four-digit subject numbers were allocated on the morning of dosing according to the code 1001 to 1020 for males and 1021 to 1040 for females using the lowest number available. Replacement subjects were allocated subject numbers 9001 to 9020 for males and 9021 to 9040 for females, where the last three digits are the same as those of the original subject (e.g., if Subject 1005 withdraws, the replacement will have the Subject Number 9005 and will receive the same regimen as Subject 1005. In addition, for the 900 mg fed cohort, subjects received the same regimen as the withdrawn subject in both the fasted and fed states.
Blinding: Pharmacy staff -An unblinded pharmacy staff was required at the Clinical Site to comply with the study's randomization and blinding requirements. At the clinical research unit, prior to study initiation, the Principal Investigator was responsible for designating a quali ed pharmacy staff to serve as the unblinded pharmacy staff in the study. Unblinded pharmacy staff could dose subjects but could not participate in any subject assessments. Throughout the study, the designated unblinded pharmacy staff was responsible for all drug accountability issues, including preparing, labeling, dispensing, and dosing study drug according to the randomization code provided, yet remain independent of all subject assessments. The pharmacy staff followed the standard operating procedures and Work Instructions related to pharmacy services and protocol-speci c requirements. Randomization codes were provided to the unblinded pharmacy staff. Con rmation of receipt of the randomization code was required by the Sponsor. The Principal Investigator was ultimately responsible for ensuring that the integrity of the blind is maintained throughout the study at the site and required to notify the Safety Review Committee in the event of any breaking of the blind for any reason. Clinical Research Staff-All observers who evaluate any reported AE, laboratory abnormalities, ECGs, and changes in the ECGs were blinded as to what treatment each subject is assigned. Study Subjects-All subjects were blinded as to which treatment they received and dose. Data Sciences Staff-The unblinded statistician was not involved in any decisions relating to populations for analysis prior to unblinding. Interim PK parameter estimations were performed using bioanalytical data applied with subject aliases to maintain the study blinded. Further, the Data Sciences department did not have access to the randomization schedule before database lock and unblinding. Pharmacovigilance -A copy of the randomization schedule was made available to the pharmacovigilance provider for analysis of pharmacovigilance. Bioanalytical Laboratory -All samples were sent to the bioanalytical laboratory for analysis. The bioanalytical laboratory was unblinded, a copy of the randomization schedule provided, and only ran the analysis on active treatment subjects. Pharmacokinetic Analysis -PK analysis was completed only on the active treatment subjects and blinded by subject aliases for interim assessments.
Check-In Procedures: At each check-in all subjects were evaluated to con rm they continue to meet all the inclusion criteria and none of the exclusion criteria. General risk mitigation against COVID-19 was implemented in accordance with Quotient Sciences' monitoring and prevention control measures. A urine sample was collected from all subjects at each study check-in to screen for drugs of abuse and alcohol. If at any time an alcohol or drug test is positive, the subject is discontinued from study participation. Female subjects of childbearing potential and male subjects will be asked to con rm that they still adhere to the contraceptive criteria. A serum pregnancy test was performed on all female subjects at each checkin using the clinical chemistry blood sample; this test had to be negative for the subject to continue study participation. Blood and urine samples were also collected at admission for clinical laboratory assessments.
Con nement: NHVs were admitted to the research center in the morning of the day before study drug administration (Day -1) to ensure a minimum 10-h fast before dose administration for the fasted regimens. Subjects enrolled in the study had remained in the research center until the completion of the study procedures scheduled for 48 h after dose administration (Day 3), and they returned to the research center for 3 outpatient visits (Days 4, 5, and 6).
Follow-Up: A follow-up phone call took place between Days 7 and 10 (+ 2 days) to ensure the ongoing well-being of the subjects. If a subject reported any AEs which could present a cause for concern, they were required to attend the clinical unit for a further follow-up assessment (as an unscheduled visit) and were followed up until the AE had resolved. Completion of the last follow-up call or unscheduled followup visit was considered the end of the study. This extraction was taken by the third-party Holter provider at the planned time, and therefore, no other procedures were performed within the resting period. Where one period of ECG rest ran into the rest period for the next ECG extraction, the minimum resting period before ECG extraction did not need to be started again except in cases where the rest was disturbed (e.g., subjects had to get up for a comfort break after the preceding extraction). Subjects were allowed to move more freely outside of the primary ECG rest periods. Comfort breaks for hygienic purposes were allowed each morning well in advance of the required supine/ECG extraction points. Where any scheduled rest times were missed, where there was less than a 10-min rest period, or where the nal 5 min of a rest period was interrupted (for example, by subject movement or loss of leads), a protocol deviation was recorded, and the Holter provider was informed.
Loss of leads or interruption of the recording outside of the scheduled rest times was not considered a protocol deviation but was recorded in the source. The acceptable deviations for ECG rest periods from the nominal time point were pre-dose and post-dose ECG resting periods ending within ± 15 min of the nominal time point. ECGs were collected electronically and over-read by cardiologists at the Central ECG Laboratory, Banook Group. The Central ECG Laboratory over-read was used for data analysis and report writing purposes.
Adverse Event: An AE was de ned as any untoward medical occurrence associated with the use of a drug in humans, whether or not considered drug-related. An AE could be any unfavorable and unintended sign (e.g., an abnormal laboratory nding), symptom, or disease temporally associated with the use of a drug, without judgment to causality. An AE could arise from any use of the drug (e.g., off-label, use in combinations with another drug) and from any route of administration, formulation, or dose, including an overdose.
Recording Adverse Events: AEs were recorded from the time of providing written informed consent until 30 days after the last dose of study drug. During each study visit, the subject was questioned directly regarding the occurrence of any adverse medical event according to the schedule in eCRF. All AEs, whether ascribed to study procedures or not, were documented immediately in the subject's eCRF. This included the date and time of onset, a description of the AE, severity, seriousness, duration, actions taken, outcome, and an Investigator's current opinion on the relationship between the study drug and the event.
A diagnosis and nal opinion on the relationship between the study drug and the event were provided at the end of the study by the Quotient Sciences Principal Investigator. Any subject who withdrew from the study due to an AE was followed up until the outcome was determined and written reports were provided by the Investigator. Single Cell Multiome ATAC + Gene Expression (GEX) sequencing data. Brie y, raw BCL les from the Illumina NovaSeq were demultiplexed into paired-end, gzip-compressed FASTQ les that were generated using cellranger-arc mkfastq and default parameters. Read alignment, ltering, barcode counting, peak calling, and counting of both ATAC and GEX molecules were performed using cellranger-arc count. The rat genome mRatBN7 and its annotated transcriptome were used as the reference for alignment. Only con dently mapped reads with valid barcodes, unique molecular identi ers (UMIs), and non-PCR duplicated were retained. The overall sequencing quality was evaluated by looking at the summary metrics of the web_summary.html le generated for each sample. Processed data sets of multiple samples were then aggregated with cellranger-arc aggr to normalize input runs to the same median fragments per cell across samples. The linkage between chromatin accessibility and GEX was also established for each sample with this tool, as the ATAC and GEX measurements are on the very same cell.

Seurat and single cell downstream Analysis
Standard pre-processing through the Seurat work ow was performed for single-cell RNAseq data.
Mitochondrial genes were identi ed, and cells expressing >5 percent of mitochondrial features were removed. The dataset was log normalized with a size factor of 10,000 molecules for each cell using the NormalizeData function. Z-score transformation was performed across all cells to standardize expression values for genes using the ScaleData function 52 . Highly variable features for each group were identi ed using the FindVariableFeatures function. The top 10 variable features were plotted.
To visualize the data principal component analysis (PCA) was performed for dimensionality reduction, PCA dimensions were further reduced into two dimensions using UMAP. Weighted shared nearest neighbor and clustering of single cells was performed by Louvain algorithm. A table of logged 2-fold change and p values between each treatment group was calculated with Seurat. This calculation determines differential expression using non-parametric Wilcoxon rank sum test; these values were used in heatmaps and dot plots showing differential expression of top 50 DE and pain-related genes.
Additionally, the top 50 differentially expressed genes were used in Gene Ontology enrichment analysis described below.
Cell type annotation was performed with the ScType package, which utilizes the largest database of Unbiassed pathways and gene networks were algorithmically generated based on their connectivity and Page 43/53 assigned a score. The score considers the number of focus genes in the network and the size of the network to approximate how relevant this network is to the original list of genes. The networks identi ed are then presented as a graph indicating the molecular relationships between genes/gene products. Upstream causal networks indicating potential/predicted upstream relationships based on the input gene list are also generated, as well as enrichment plots based on the most signi cant/highest-scoring pathways containing genes from the uploaded dataset. The "path designer" module was used to polish the network images. Data from IPA for canonical pathway enrichment was exported, and histograms were produced using RStudio 2021.09.0.
Tables Table 1 is available in the Supplementary Files section. Figure 1 SRP-001's absent hepatotoxicity is due to lack of NAPQI formation and maintenance of hepatic tight junction integrity. a, Nitrotyrosine-labeled hepatic sections from CD1 mice (100x, a, rst column) demonstrate centrilobular hepatic necrosis following per os toxic doses (600 mg/kg) of ApAP, but not SRP-001 [(n=5) mice per treatment group]. Hepatic tight junctions (ZO-1 label [3D-stacking, 1000x] a, second column) present between hepatocytes reveal a clear "chicken wire" con guration (vehicle) and are Analgesia is mediated via AM404 production in the PAG region and human Single Ascending Dose (SAD) Phase 1 clinical trial reveals a favorable pharmacokinetic pro le. a, Detection of deuterated (D 10 -SRP-001) in the rat brain 30 min after IP injection. The full fragmentation spectrum of D 10 -SRP-001 detected in the brain (red) matches b, the standard SRP-001 compound spectrum (blue). c, The structure of D 10 -SRP-001 shows fragmentation patterns of D 10 -SRP-001 that match the spectra shown in the brain (red) and

Figures
with the deuterated standard (blue). d, The MRM (m/z 416 → 186) for D 10 -SRP-001 shows the elution time at ~2.2 min. e, Quanti cation of AM404 production in the CNS periaqueductal gray region. The highest AM404 is produced in animals treated with CFA and dosed with SRP-001, followed by rats injected with vehicle (0.9% saline) and SRP-001. AM404 production is lower in the periaqueductal gray region of rats injected with CFA and dosed with ApAP, and there is almost no AM404 detected in the periaqueductal gray region of rats injected with either CFA alone or vehicle. f, AM404 is expressed in the periaqueductal gray area of the brain following SRP-001 IP injection. LC-MS/MS detection of AM404 in the rat periaqueductal gray area after SRP-001 (top panel) and ApAP (bottom panel) IP injections. The AM404 structure and major fragments. g, The peaks are con rmed to be AM404 by co-spiking AM404 standard to the samples (red and orange, respectively). The peaks in the AM404 standard (top panel, pure AM404 analytical standard (10 ng/mL, Cayman Chemical, Ann Arbor, MI.) and SRP-001 from the periaqueductal gray (bottom panel, SRP-001) 32 mg/kg IP injection and animals sacri ced 30 mins after injection (same as in the CFA/von Frey analgesia model). From the whole brain, the periaqueductal gray area was excised. The major peaks detected by LC-MS/MS demonstrate that the peak 9.9 min from LC is the AM404 compound. These peaks align to the e, AM404 fragments. h, Design of the rst-in-human Phase 1 clinical trial for SRP-001. A randomized, double-blind, placebo-controlled study to assess the safety, tolerability, and pharmacokinetics of single ascending oral doses of SRP-001 and to characterize the effect of food on the pharmacokinetics of SRP-001 in healthy male and female subjects. Single ascending dose (SAD) escalation from 300 mg to 2,000 mg (fasted state) and 900 mg (fed state). i, Geometric mean SRP-001 plasma concentration-time pro les following oral administration of SRP-001 (logarithmic plot) show a proportional or super-proportional increase in Cmax (peak time to concentration at 1 h post-dose) with a mono-or bi-phasic concentration decline with a geometric mean T 1/2 of 4.1 h.

Figure 4
Single-cell transcriptomics of midbrain periaqueductal gray (PAG) cells in CFA-induced chronic in ammatory pain model. a, Overview of single-cell RNA sequencing (scRNA-seq) experimental design to de ne the mechanism of action (MOA) of ApAP and SRP-001. Left hind paws of Sprague-Dawley rats were injected with complete Freund's adjuvant (CFA) to induce local in ammation and a chronic pain state, or vehicle (0.9% saline). They were then dosed with either ApAP or SRP-001 (100 mg/kg). Periaqueductal gray (PAG) midbrain region was dissected to isolate cell nuclei for sequencing library generation using the 10x Genomics Chromium platform. Single cell data were embedded after dimensionality reduction using uniform manifold approximation and projection (UMAP). Cell clusters were annotated, followed by differential gene expression analysis. b, UMAP plots showing the distribution of annotated cell clusters generated by the work ow of Seurat across 4 different samples -Vehicle, CFA_Veh, CFA_ApAP, and CFA_SRP-001, respectively. c, Heatmap of top 40 canonical pathways for neuronal clusters across samples -CFA_Veh, CFA_ApAP, and CFA_SRP-001 with the comparisons between them as follows -Vehicle vs CFA_Vehicle, CFA_Vehicle vs CFA_ApAP, and CFA_ApAP vs CFA_SRP-001. The pathways shown in the heatmap were predicted using Qiagen Ingenuity Pathway Analysis (IPA) tool from the normalized matrix after comparing the differentially expressed genes between the neuronal clusters of the samples that were compared against each other. The heatmap shows that when CFA_Vehicle is compared to Vehicle, there is upregulation of several genes that are predicted to affect cellular signaling related to these pathways highlighted in the heatmap, and when CFA_ApAP and CFA_SRP-001 are compared to CFA_Vehicle, there is downregulation of those upregulated pathways in both cases, with more downregulation for CFA_SRP-001 than CFA_ApAP. Similarly, there is predicted downregulation of mitochondrial dysfunction and Granzyme A signaling pathways for CFA_Vehicle, which are restored by CFA_ApAP and CFA_SRP-001 with CFA_SRP-001downregulating more based on the color intensity of the heatmap.